Fatty acids of plants are important components that make cell membranes and seed oils. In particular, microsomal oleic acid desaturase, which is present in the endoplasmic reticulum of plant cells, is an enzyme that catalyzes the conversion of monounsaturated fatty acid, oleic acid, at the sn-1 and sn-2 positions of phosphatidylcholine, into diunsaturated fatty acid, linoleic acid.
There have been reports about presence of genomic genes (FAD2) encoding the microsomal oleic acid desaturase in Arabidopsis, Petunia, and cotton. According to the reports, while one FAD2 gene is present on the genome of Arabidopsis, two or more FAD2 genes are present on each genome of Petunia and cotton [Okuley et al., 1994, Plant Cell; Verwoert et al., 2000, Biochemistry Society Transactions; Pirtie et al., 2001, Biochimica et Biophysica Acta]. In particular, it has been known that when two or more FAD2 genes are present on a plant genome, at least one is involved in production of linoleic acid that accumulates in a seed oil. Until now, while the presence of the FAD2 gene in the above-mentioned three plants has been reported, no studies on the functional analysis of its promoter have been reported.
Recently, many attempts have been made to improve genetic characteristics of plants using genetic engineering technology. In this respect, expression of a useful foreign gene in a transgenic plant requires a promoter for the gene expression. For this, conventionally, a promoter derived from a cauliflower mosaic virus (CaMV35S) gene that induces gene expression at all tissues of a plant has been widely used. However, this promoter cannot induce seed-specific gene expression. Meanwhile, there have been reports about promoters that induce tissue-specific expression, for example, seed-specific expression [Plant Cell Technology, 1991, 3: 568-576], leaf- or flower-specific expression [Science, 1990, 250: 931-936], and root-specific expression [Plant Cell Technology, 1991, 3: 577-587]. However, the above seed-specific expression promoter induces gene expression during whole stages for seed development, not during a specific development stage.
In view of these problems, based on the fact that the microsomal oleic acid desaturase derived from sesame is specifically expressed during a seed development stage, the present inventors cloned the genomic gene encoding microsomal oleic acid desaturase and its promoter, inserted the promoter into a binary vector and a transient expression vector, and then introduced the vectors into the seeds of model plants, Arabidopsis and sesame. As a result, the present inventors found that the sesame microsomal oleic acid desaturase (Si-FAD2) promoter is a novel promoter that induces the expression of a foreign gene in a seed development stage-specific manner, and completed the invention.
Furthermore, a 127 bp active fragment of the promoter essential for the seed-specific expression has been identified. Also, the present inventors found that when the promoter is used together with an intron present on the Si-FAD2 gene, the expression level of a foreign gene in a seed is enhanced by at least 40-fold, and completed the invention. In this regard, the intron is useful in high expression of a useful foreign gene.
Therefore, the present invention provides a seed-specific, in particular, a seed development stage-specific expression promoter derived from the Si-FAD2 gene and a 127 bp active fragment thereof.
The present invention also provides an intron that enhances the expression level of a foreign gene under the control of the above-described seed-specific expression promoter.
The present invention also provides a seed-specific expression vector containing the above-described promoter and/or intron and a transgenic plant transformed with the seed-specific expression vector.
The present invention also provides a method for expressing a foreign gene in a transgenic plant using the above promoter. This method is useful in producing a useful product in a seed development stage-specific manner or functionally modifying conventional product in seeds.